Since the discovery that nitric oxide (NO) is identical to the elusive endothelium-derived relaxing factor [1], many more profound biological roles of NO have been identified and elucidated [2-6]. These findings prompted further exploration of potential applications of exogenous NO in wound healing, cardiovascular diseases, respiratory diseases, cancer therapy, nerve system reconstruction, as well as new functional medical devices. In this regarding, local delivery of NO has great potential in gaining clinical utility as evident in its demonstrated success in treating wound infection using topical applied NO gas [7]. However, the short half life of this small gaseous molecule and its intrinsic instability have presented great challenges for its incorporation into pharmaceutical dosage forms and drug delivery systems. It has been reported that NO endogenously synthesized by vascular endothelial cells has a very short biological half life of 5 sec or less [8, 9]. Because NO is rapidly scavenged by hemoglobin, its site of action in the tissue would be localized to where it is generated. The chemical instability of NO in cells and tissue has been attributed to its rapid oxidation to both NO2− and NO3−.
Besides organic nitrates and sodium nitrite which are well known sources of NO, there are two other families of NO precursors which have been studied extensively. One consists of diazeniumdiolates and the other S-nitrosothiols. Diazeniumdiolates include compounds of structure R1R2NN(O)═NOR3, which are also known as NONOates. Numerous efforts have been made in developing NO-releasing materials based on this class of NO donors [10, 11]. These include the incorporation of diazeniumdiolates into different polymeric matrices through either physical blends or covalent attachment to the polymer backbone or side chains. Related prior art approaches on diazeniumdiolates are described below.
In WO 2005/011575, WO 2005/07008, and WO 2006/058318, Smith disclosed NO releasing devices based on either ion exchange resins or polyethyleneimine (PEI) fibrous multilaminates in which diazeniumdiolate moieties are attached to the polymer matrix through either ionic or covalent bonding. Upon contacting such NO derived polymers with an activator such as water, hydrogen cation or ascorbic acid at the time of activation or application to the wound, local NO release can be generated. However, the duration of NO release from such systems is short, typically lasting only 0.5 to 3 hours from the ion exchange resin systems and at most one to two days from the fibrous multilaminate devices.
Meyerhoff and coworkers disclosed in U.S. Pat. No. 6,841,166 and US 2006/0008529, NO releasing polymeric materials for thromboresistant blood contacting devices based on hydrophobic polymers (such as silicone rubber, poly(vinyl chloride), polyurethanes, etc.) containing a discrete NO doner including diazeniumdiolate derivatized fumed silica, dispersed diazeniumdiolates or covalently linked diazeniumdiolates, together with an acidic activator and a plasticizer. During activation, water penetrates slowly into the hydrophobic polymer matrix resulting in a prolonged release of NO into the aqueous environment up to several days. These systems have also been tested as implantable grafts, catheters or coatings on biomedical devices for the delivery of NO for the treatment of cardiovascular restenosis and blood circulation disorders [12-15]. In addition to biocompatibility concerns, these extremely hydrophobic materials are not suitable for wound healing applications because of their poor water absorbency and poor bioadhesion at the wound site.
Moreover, one major limitation in the in vivo application of this class of NONOate donors is the potential toxicity of leachable diazeniumdiolates and their decomposition products, particularly nitrosoamines, as elucidated in U.S. Pat. No. 6,841,166. Prior art approaches mentioned above as well as in U.S. Pat. No. 6,703,046 had employed hydrophobic polymers to minimize such leaching. However, leaching can still occur from these polymers containing hydrophilic acidic additives and plasticizers. Additionally, one established diazeniumdiolate pro-drug, V-PYRRO/NO, has the potential of forming N-nitrosopyrrolidine, which is one of the most potent experimental hepatocarcinogens known [16]. Furthermore, diamine-based and polyethylenimine-based diazeniumdiolates released into aqueous medium have been shown to form measurable levels of nitrosamines, a known class of carcinogens [12]. Therefore the application of diazeniumdiolates in vivo, especially for wound healing, appears to be limited.
Another major class of NO donors is S-nitrosothiols, which are compounds having the generic structure of R—SNO. As important endogenous and exogenous sources of NO, RSNOs are widely distributed in vivo and have been shown to store, transport, and release nitric oxide in the mammalian body [17]. In addition, their ability to generate NO upon aqueous activation in physiological fluid is particularly advantageous for the local delivery of NO, targeting only to a specific tissue without having to achieve a systemic load. Among the various endogenous RSNOs, S-nitrosoglutathione (GSNO) has attracted significant attention due to its ease of synthesis through a spontaneous reaction between glutathione and sodium nitrite at room temperature and its ability to be isolated as a solid, [18]. However, the stability of these small molecular RSNOs is less than satisfactory as the S—NO bond is both thermally and photolytically labile, and susceptible to hemolytic cleavage leading to the spontaneous release of NO and its rapid inactivation, thus limiting their suitability for practical applications including wound healing.
de Oliveira and coworkers have physically incorporated S-nitrosoglutathione (GSNO) and/or S-nitroso-N-acetyl-cysteine (SNAC) into films and gels based on water soluble polymers, such as poly(vinyl alcohol), poly(vinyl pyrrolidone, or Pluoronic F127 hydrogel, for transdermal NO delivery [19-22]. Their animal results show that repeated topical application of GSNO-containing hydrogel during the early phases of rat cutaneous wound repair accelerates wound closure and re-epithelialization [23]. However, a prolonged NO release would be more desirable from a patient compliance point view in order to avoid repeated applications.
Katsumi and co-workers synthesized a macromolecular carrier for S-nitrosothiol based on bovine scrum albumin (BSA) and poly(ethylene glycol) (PEG)-conjugated BSA by covalently attaching nitrite to cysteine residues on BSA [24, 25]. Similarly, West et al demonstrated in U.S. Pat. No. 7,052,711 that S-nitrosocysteine (CysNO) immobilized within a poly(ethylene glycol) hydrogel reduced platelet adhesion and smooth muscle cell proliferation in in vitro cell culture. However, these reported hydrophilic systems lack the desired stability as the S—NO bond is both thermally and photolytically labile, and susceptible to hemolytic cleavage leading to the spontaneous release of NO and its rapid inactivation. As a result, the nitric oxide release duration from compounds of the prior art cannot be maintained for any extended period, which is, generally, not more than several hours.
Prior art methods of physically mixing GSNO in a polymer [21-24] to form an admixture and mixing a NO precursor with an activator to generate GSNO, either in situ at the time of application as described in WO2006/095193 or in vitro prior to its application to wounds as described in WO2008/031182, do not address the issue of short half-life of GSNO, because once GSNO is formed or released, it is still susceptible to degradation due to heat, moisture and light. In fact, in most of these prior art approaches, the release of NO or GSNO, is usually very rapid and lasts no more than several hours thus necessitates repeated application.
There is, therefore, a need in the art for achieving a stable NO delivery system that provides controllable and durable release of NO for wound healing applications.